Sustained-release nanoparticle compositions and methods for using the same

ABSTRACT

The present invention is a composition composed of a therapeutic agent encapsulated in a copolymer of an N-alkylacrylamide, a vinyl monomer, and a polyethylene glycol (PEG) conjugate and a method for using the same in the treatment or prevention of a disease or condition.

BACKGROUND OF THE INVENTION

Restenosis is a complex process which is believed to be triggered byblood vessel wall injury following an intervention to relieve anarterial obstruction (e.g., angioplasty, atherectomy, or stenting).Mechanisms contributing to restenosis include elastic recoil, smoothmuscle cell migration and proliferation, enhanced extracellular matrixsynthesis vessel wall remodeling, and thrombus formation (Haudenschild(1993) Am. J. Med. 94:40S-44S; Lovqvist, et al. (1994) J. Intern. Med.233:215-226; Koster, et al. (1995) Angiology 46:99-106; Wilcox (1991)Circulation 84:432-435; Wilcox (1993) Am. J. Cardiol. 72:88E-95E; Wilcoxand Blumenthal (1995) J. Nutr. 125:631S-638S). Restenosis after aninitial successful angioplasty of an atherosclerotic plaque remains themajor limitation of coronary angioplasty in humans.

Therapeutic approaches for the prevention of restenosis have focused oneither intervening in early events, such as platelet deposition orthrombus formation, or preventing later events, i.e., proliferation ofsmooth muscle cells and matrix formation. Several classes of therapeuticagents have been used experimentally in animal studies. These haveincluded anticoagulants, anti-inflammatory drugs, anti-platelet agentswhich can block initial events, and antiproliferative agents whichinhibit the later events in the pathogenesis of restenosis (Herrman, etal. (1993). Drugs 46:18-52, 249-262; Marmur, et al. (1994) J. Am. Coll.Cardiol. 24:1484-1491; Mathias (1991) Semin. Thromb. Hemostat 17:14-20).Other approaches to treat restenosis have involved the use of antisenseoligonucleotides to block transcription of certain cytokines orproto-oncogenes, such as c-myc or c-myb (Wilcox (1993) supra; Bennett,et al. (1994) J. Clin. Invest. 93:820-828; Epstein, et al. (1993)Circulation 88:1351-1353; Edelman, et al. (1995) Circ. Res. 76:176-182)Gene therapy strategies have also been investigated (Wilcox (1993)supra; Muller (1994) Br. Heart J. 72:309-312; Nabel, et al. (1990)Science 249:1285-1288; Nabel (1995) Cardiovasc. Res. 28:445-455;Bennett, et al. (1994) supra; Epstein, et al. (1993) supra; Edelman, etal. (1995) supra; Feldman and Isner (1995) J. Am. Coll. Cardiol.26:826-835).

Modification of the restenosis process by conventional pharmacologic ormechanical approaches (e.g., stenting) (Wilensky, et al. (1993) TrendsCardiovasc. Med. 3:163-170) have been used in the clinical setting. Drugtherapies have included antiplatelet and anticoagulant agents, calciumchannel antagonists, inhibitors of angiotensin converting enzyme,corticosteroids, and fish oil diet (Herrman, et al. (1993) supra).Mechanical approaches include deployment of metallic or polymeric stentsin the artery to inhibit elastic recoiling which usually occurs withinhours following angioplastic procedure and results in renarrowing of theartery lumen (Herrman, et al. (1993) supra; De Scheerder, et al. (1995)Atherosclerosis 114:105-114; De Foley, et al. (1993) Am. Heart J.125:686-694; Kuntz, et al. (1993) J. Am. Coll. Cardiol. 21:15-25;Lambert, et al. (1994) Circulation 90:1003-1011; Mitchel and McKay(1995) Cathet. Cardiovasc. Diagn. 34:149-154; Buchwald, et al. (1993) J.Am. Coll. Cardiol. 21:249-254). Other approaches include atherectomy,local treatment of arterial lesions with laser, thermal energy, and β-and γ-radiations following interventional procedures (Buchwald, et al.(1992) Am. Heart J. 123:878-885; Kouek, et al. (1992) Circulation86:1249-1256; Israel, et al. (1991) J. Am. Coll. Cardiol. 18:1118-1119).

Administration of therapeutic agents at the site of arterial injuryrather than by systemic administration has been discussed (Labhasetwar,et al. (1997) Adv. Drug Del. Rev. 24:63-85). Experimental studies inanimal models of restenosis have been used to investigate local deliveryof therapeutics for the prevention of restenosis (Lambert, et al. (1994)supra; Garcia, et al. (1990) Surg. Gynecol. Obstet. 171:201-205;Edelman, et al. (1990) Proc. Nat. Acad. Sci. USA 87:3773-3777; Edelman,et al. (1993) Proc. Nat. Acad. Sci. USA 90:1513-1517; Edelman andKarnovsky (1994) Circulation 89:770-776; Nathan, et al. (1995) Proc.Nat. Acad. Sci. USA 92:8130-8134; Okada, et al. (1989) Neurosurgery25:892-898; Villa, et al. (1994) J. Clin. Invest. 93:1243-1249; Villa,et al. (1995) Circ. Res. 76:505-513). Adventitial drug implants(Edelman, et al. (1990) supra; Villa, et al. (1994) supra; Simons, etal. (1992) Nature 359:67-70; Simons, et al. (1994) J. Clin. Invest.93:2351-2356), stents (Lincoff, et al. (1994) J. Am. Coll. Cardiol.23:18A; Jeong, et al. (1994) Circulation 92:137), and catheter-baseddelivery systems (Steg, et al. (1994) Circulation 90:1648-1656;Fernandez, et al. (1994) Circulation 89:1518-1522) have been disclosed.Further, Lanza, et al. ((2002) Circulation 106:2842) teach targetedparamagnetic nanoparticles containing paclitaxel for the prevention ofrestenosis after angioplasty.

U.S. patent application Ser. No. 09/847,945 teaches methods for treatinghyperplasia in a subject by delivering at least one drug in nanoparticleform and dispersed in a biocompatible protein. This reference disclosesthe use of paclitaxel, rapamycin, steroids, and the like, as suitablecandidates to inhibit proliferation and migration of cells. Thisreference does not teach block co-polymer nanoparticles.

U.S. Pat. No. 6,322,817 teaches a pharmaceutical formulation ofpaclitaxel, wherein the paclitaxel is entrapped into nanoparticlescomprising at least one type of amphiphilic monomer which is polymerizedby adding an aqueous solution of cross-linking agent. This referencediscloses a preferred combination of amphiphilic monomers comprisingvinyl pyrrolidone, N-isopropylacrylamide, and monoester of polyethyleneglycol maleic anhydride cross-linked with a bi-functional vinylderivative such as N,N′-methylene bis-acrylamide useful in the treatmentof pathological conditions arising out of excessive proliferation ofcells such as rheumatoid arthritis or cancer.

U.S. Pat. No. 6,759,431 discloses methods for treating or preventingdiseases associated with body passageways by delivering to an externalportion of the body passageway a therapeutic agent such as paclitaxel,or an analogue or derivative thereof encapsulated in polymeric carriers.Intravenous or oral delivery of agents for preventing disease orconditions is generally ineffective because these routes of delivery donot provide a therapeutic dose of the agent to the target site for aprolonged period of time. Therefore, there is a need in the art forsite-specific therapeutics to prevent the localized pathophysiologicprocess of select disease or conditions. The present invention meetsthis long-felt need.

SUMMARY OF THE INVENTION

The present invention is a sustained-release nanoparticle compositioncomposed of a copolymer of an N-alkylacrylamide, a vinyl monomer, and apolyethylene glycol conjugate. In one embodiment, the N-alkylacrylamide,vinyl monomer, and polyethylene glycol conjugate are in a ratio of70-90:9-20:1-10. In another embodiment, the N-alkylacrylamide comprisesN-methyl-N-n-propylacrylamide, N-methyl-N-isopropylacrylamide,N-propylmethacrylamide, N-isopropylacrylamide, N,n-diethylacrylamide,N-isopropylmethacrylamide, N-cyclopropylacrylamide,N-ethylmethyacrylamide, N-methyl-N-ethylacrylamide,N-cyclopropylmethacrylamide, or N-ethylacrylamide. In a furtherembodiment, the vinyl monomer comprises a vinyl alcohol, a vinyl ether,a vinyl ester, a vinyl halide, a vinyl acetate, or a vinyl pyrrolidone.In yet a further embodiment, the polyethylene glycol conjugate comprisesPEGylated maleic acid, PEGylated vinylsulfone, PEGylated iodoacetamideor PEGylated orthopyridyl disulfide. In particular embodiments, thesustained-release nanoparticle composition further contains atherapeutic agent such as an antibiotic, anti-restenotic agent,anti-proliferative agent, anti-neoplastic, chemotherapeutic agent,cardiovascular agent, anti-inflammatory agent, immunosuppressive agent,or anti-tissue damage agent. Such nanoparticle compositions generallyhave a diameter in the range of 20 nm to 100 nm and are used locally forthe prevention or treatment of diseases or conditions.

The present invention is also a method for using a sustained-releasenanoparticle composition for preventing or treating a disease orcondition. The method involves locally administering an effective amountof a sustained-release nanoparticle composition containing a therapeuticagent to a patient having or at risk of having a disease or conditionthereby preventing or treating the disease or condition in the patient.In particular embodiments, the patient is at risk of restenosis, i.e.,the patient has undergone angioplasty, atherectomy, or stenting.

DETAILED DESCRIPTION OF THE INVENTION

It has now been appreciated that a therapeutic agent encapsulated in ananoparticle composed of a copolymer of an N-alkylacrylamide, a vinylmonomer, and a polyethylene glycol (PEG) conjugate can be administeredlocally to effectively deliver high concentrations of the therapeuticagent. In particular, improved loading efficiency of the therapeuticagent into nanoparticles has been achieved with higher molar ratios ofthe N-alkylacrylamide component of the nanoparticle. Therapeutic agentsentrapped in the nanoparticles disclosed herein are released slowly asthe nanoparticles dissociate, thus providing sustained drug releasecharacteristics. A nanoparticle of the present invention serves as aneffective drug carrier for intraluminal drug delivery because of itsnanometer size range that results in better drug uptake and penetrationin the arterial wall than that of a larger drug carrier system such as amicroparticle or liposome.

By way of illustration, a specific therapeutic agent-nanoparticlecomposition was prepared to demonstrate sustained-release,biocompatibility, arterial localization, inhibition of cellproliferation, and prevention of hyperplasia in a rat carotid arterymodel of restenosis. The illustrative composition consisted ofrapamycin, a potent macrolide antibiotic which is known to inhibitproliferation and migration of vascular smooth muscle cells (VSMCs), andpolymeric nanoparticles synthesized using a copolymer ofN-isopropylacrylamide (NIPAM), vinyl pyrrolidone (VP), and PEGylatedmaleic acid (PEGMA) (80:15:5) which were cross-linked withN,N′-methylene bis-acrylamide (MBA). When compared to nanoparticleslacking PEG, the addition of PEG to the nanoparticle composition wasfound to provide greater stability to the nanoparticles, decreasedaggregation and increased drug loading. On a weight per weight basis,nanoparticles lacking a PEG conjugate (i.e., containing NIPAM:VP, 80:20)incorporated 2.5% of rapamycin. In contrast, preformed nanoparticlescontaining a PEG conjugate (i.e., NIPAM:VP:PEGMA, 80:15:5) incorporatedup to 4.5% of rapamycin.

Rapamycin-loaded nanoparticles exhibited sustained-release of the loadeddrug under in vitro conditions. The release rate was high during theinitial phase and decreased exponentially with time (Table 1).

TABLE 1 Days Cumulative % Release of Rapamycin (±SEM) 0.5  3.81 (±0.18)1 20.05 (±0.45) 2 33.81 (±1.67) 4 49.39 (±1.43) 6 60.32 (±2.28) 10 67.26(±1.20) 14 73.77 (±1.78) 21 79.35 (±1.94) 28 84.36 (±2.15)

To demonstrate biocompatibility, vascular smooth muscle cells wereexposed for 48 hours to various concentrations of nanoparticles lackingrapamycin (0, 10, 50, 100, and 1000 μg/mL) and cell viability wasdetermined using a standard MTS assay. The nanoparticles exhibited notoxic effect to vascular smooth muscle cells in vitro up to a dose of1000 μg/mL.

To demonstrate the anti-proliferative effects of rapamycin-loadednanoparticles on vascular smooth muscle cells, cells were incubated withvarious concentrations (1, 10, 100, 1000 ng/mL) of rapamycin either insolution (i.e., dissolved in methanol and diluted in cell culturemedium) or loaded in nanoparticles. Proliferation was measured using anMTS assay. Although rapamycin in solution and rapamycin-loadednanoparticles exhibited a similar dose-dependent inhibition of vascularsmooth muscle cell proliferation, inhibition with rapamycin-loadednanoparticles was significantly greater at later time points. Forexample, rapamycin (1 ng/mL) in solution and in nanoparticles showedsimilar inhibition at 5 days; however, at 8 days, rapamycin innanoparticles demonstrated 20% more inhibition than rapamycin insolution (Table 2). Therefore, rapamycin-loaded nanoparticlesdemonstrate sustained inhibition of vascular smooth muscle cellproliferation.

TABLE 2 Cell Viability (Absorbance ± SEM) Treatment Day 2 Day 5 Day 8Medium 0.332 ± 0.010 0.748 ± 0.048 1.065 ± 0.068 0.357 ± 0.047 0.758 ±0.049 1.075 ± 0.061 0.343 ± 0.029 0.758 ± 0.049 1.087 ± 0.077 0.344 ±0.026 0.773 ± 0.035 1.059 ± 0.049 Control 0.316 ± 0.027 0.712 ± 0.0541.016 ± .031  Nanoparticle 0.346 ± 0.019 0.705 ± 0.039 1.050 ± 0.0210.354 ± 0.015 0.707 ± 0.039 1.050 ± 0.021 0.419 ± 0.028 0.699 ± 0.0311.017 ± 0.025 Rapamycin in Solution   1 ng/mL 0.177 ± 0.017 0.363 ±0.019 0.633 ± 0.019  10 ng/mL 0.199 ± 0.023 0.369 ± 0.036 0.688 ± 0.040 100 ng/mL 0.195 ± 0.014 0.325 ± 0.023 0.692 ± 0.040 1000 ng/mL 0.196 ±0.011 0.354 ± 0.015 0.693 ± 0.058 Rapamycin-loaded Nanoparticle   1ng/mL 0.184 ± 0.012 0.364 ± 0.027 0.489 ± 0.044  10 ng/mL 0.196 ± 0.0270.368 ± 0.029 0.431 ± 0.037  100 ng/mL 0.178 ± 0.011 0.326 ± 0.019 0.402± 0.020 1000 ng/mL 0.196 ± 0.007 0.362 ± 0.069 0.485 ± 0.027

The effect of rapamycin-loaded nanoparticles on the cell cycle wasdetermined by flow cytometry analysis of DNA in vascular smooth musclecells. Flow cytometry data demonstrated that the anti-proliferativeeffect of rapamycin was primarily due to inhibition of cell-cycleprogression at G1 checkpoint; the percentage of cells in G0-G1 phase was74.6% for the rapamycin-loaded nanoparticle-treated cells compared to62.7% in the untreated group. Similarly, there was a lower percentage ofcells in the proliferative S phase in the treatment group as compared tothat in the control (13.5% vs. 24.25%). (Table 3).

TABLE 3 Treatment G₀/G₁ S G₂/M % Apoptosis Medium 65.70 21.25 13.05 0.07Control Nanoparticle 67.96 19.35 12.67 0.12 Rapamycin-Loaded 74.56 13.5311.91 0.06 Nanoparticle Rapamycin in Solution 73.48 15.41 11.12 0.06

The efficacy of rapamycin-loaded nanoparticles was demonstrated in a ratcarotid artery model of restenosis. Morphometric analysis of arterialsections demonstrated significantly reduced intima to media (I/M) ratiowith localized delivery of rapamycin-loaded nanoparticles compared tocontrol nanoparticles (I/M=1.60±0.03 vs. 3.15±0.10; P<0.006)(Table 4).Intraperitoneal administration of the same dose of rapamycin-loadednanoparticles demonstrated a marginal effect on inhibition of restenosisas compared to control (I/M=2.8±0.11 vs. 3.15±0.10; P<0.006), indicatingthat the inhibitory effect was primarily due to localized delivery ofrapamycin. Inhibition of hyperplasia resulted in increased lumendiameter in locally delivered rapamycin-loaded micellar nanosystem ascompared to other controls (local rapamycin-loaded nanoparticle group,0.29±0.002 mm²; intraperitoneal rapamycin-loaded nanoparticles group,0.14±0.009 mm²; local non-drug void nanoparticles group, 0.17±0.003 mm²;P<0.006).

TABLE 4 Cross-Sectional Intima/Media Ratio Area of Lumen Treatment (mean± SEM) (mm² ± SEM) Uninjured Artery — 0.368 ± 0.012  ControlNanoparticles 3.15 ± 0.10 0.17 ± 0.002 Rapamycin-Loaded 2.87 ± 0.11 0.14± 0.009 Nanoparticles (I.P.) Rapamycin-Loaded 1.60 ± 0.03 0.29 ± 0.002Nanoparticles (Local)

Immunohistochemical staining with anti-SM antibody against α-actin(α-SMA) showed greater expression of SMA positive cells in the neointimaand adventitia of the arteries of the control group as compared to thatin the rapamycin-treated group. Immunohistochemical staining withanti-PCNA antibody showed a significantly greater number of PCNApositive cells in the neointima and adventitia in control than in thetreatment group. These results indicated that rapamycin deliverysuppressed the proliferation of VSMCs. The arterials sections in thetreatment group demonstrated significantly greater re-endothelization ofthe injured artery as compared to control (82% vs. 28%).

Further, nanoparticle localization studies were conducted using6-coumarin fluorescent dye-loaded nanoparticles and rapamycin-loadednanoparticles. Of the two carotid arteries, only one artery was injuredand infused with nanoparticles. Rapamycin- and dye-loaded nanoparticleswere found to localize in the arterial wall of the artery infused withnanoparticles, not the contra-lateral artery. Confocal microscopyanalysis of the arterial sections demonstrated localization ofnanoparticles in all the layers (intima, media and adventitia) at 1hour; however, at 24 hours the overall fluorescence activity was reducedbut was greater in the tunica media than in the intimal layer of thearterial wall. When quantified, 1.5±0.06 μg of rapamycin was present permilligram of artery at one hour after administration (Table 5). Norapamycin was detected in the non-injured contra-lateral carotid artery.Therefore, based upon the amount of rapamycin present in a 10-15 mmsegment of artery (3.2 to 4 mg of tissue), and the amount of drugadministered, the efficiency of rapamycin uptake in the target arterywas 9.1% when delivered locally via nanoparticles.

TABLE 5 Time after Rapamycin (μg/mg Administration tissue) (±SEM) 1 hour 1.5 (±0.06) 1 day 0.12 (±0.01) 3 days 0.06 (±0.03) 7 days 0.05 (±0.01)

Having appreciated the utility of a therapeutic agent encapsulated in ananoparticle composed of an N-alkylacrylamide, a vinyl monomer, and aPEG conjugate for local delivery and prevention of a condition such asrestenosis, the present invention is a sustained-release (i.e., morethan 2 to 3 weeks) nanoparticle composition and a method for using thesame for the prevention or treatment of a disease or condition.

Given the improved loading efficiency associated with higher molarratios of the N-alkylacrylamide component of the nanoparticle, oneembodiment of the present invention encompasses molar ratios of theN-alkylacrylamide, vinyl monomer, and polyethylene glycol conjugate inthe range of 70-90:9-20:1-10, respectively. In another embodiment, themolar ratios of N-alkylacrylamide, vinyl monomer, and polyethyleneglycol conjugate are in the range of 75-85:12-18:2-8, respectively. In aparticular embodiment, the molar ratio of the N-alkylacrylamide, vinylmonomer, and polyethylene glycol conjugate are desirably 80:15:5,respectively.

As used herein, an N-alkylacrylamide is a hydrophobic monomer having analkyl group of C₃ to C₆. By way of example, an N-alkylacrylamide can beN-methyl-N-n-propylacrylamide, N-methyl-N-isopropylacrylamide,N-propylmethacrylamide, N-isopropylacrylamide, N,n-diethylacrylamide,N-isopropylmethacrylamide, N-cyclopropylacrylamide,N-ethylmethyacrylamide, N-methyl-N-ethylacrylamide,N-cyclopropylmethacrylamide, N-ethylacrylamide, or the like.

A vinyl monomer used in the context of the present invention is ahydrophilic monomer having a relatively high molecular weight (e.g., inthe range of approximately 100,000 to 2,000,000, more typically in therange of approximately 500,000 to 1,500,000). Suitable vinyl monomersinclude, but are not limited to vinyl alcohol, vinyl ether, vinyl ester,vinyl halide, vinyl acetate, vinyl pyrrolidone, or copolymers thereof.

Polyethylene glycol conjugates and methods for preparing the same arewell-known in the art (Roberts, et al. (2002) Adv. Drug Deliv. Rev.54:459-476) and it is contemplated that any suitable conjugate can beused in the nanoparticles of the instant invention. In general, the PEGmoiety of the PEG conjugate is a linear compound having a molecularweight in the range of 2,000 to 50,000. It is contemplated that any PEGmoiety can be used; however, the molecular weight of the PEG moietydirectly influences the size of the resulting nanoparticle (i.e., thehigher the molecular weight, the larger the diameter of thenanoparticle). For example, it was found that the addition of a PEGconjugate, having a PEG moiety with a molecular weight of 5000, to ananoparticle composed of NIPAM and VP increased the diameter of theresulting nanoparticle by 5-10 nm when compared to the diameter of aNIPAM/VP nanoparticle lacking a PEG conjugate. Accordingly, in oneembodiment, the PEG moiety of the PEG conjugate has a molecular weightin the range of 3,000 to 10,000. In another embodiment, the PEG moietyof the PEG conjugate has a molecular weight in the range of 4,000 to7,000. In a particular embodiment, the PEG moiety of the PEG conjugatehas a molecular weight of 5,000. Particularly suitable PEG conjugatesinclude, by way of example, PEGylated maleic acid, vinylsulfone,iodoacetamide or orthopyridyl disulfide.

While the selected cross-linking agent used is not crucial, suitablecross-linking agents for use in producing the nanoparticles of thepresent invention include, but are not limited to, N,N′-methylenebis-acrylamide or N,N′-cystamine bis-acrylamide.

The biodegradable nanoparticles of the present invention can be preparedby mixing the monomers indicated herein in the presence of across-linking agent and polymerizing the mixture by free radicalpolymerization reaction using an initiator (e.g., ammonium persulfate,benzoyl perozide, or AIBN (2,2′-azo bisisobutyronitrile)). Thehydrophobic moieties of the resulting polymeric chains remain buriedinside the nanoparticles which help dissolution of drug and thehydrophilic moieties are extended outside the surface of thenanoparticles. These biodegradable nanoparticles have an averagediameter of 20 nm to 100 nm and are particularly suitable for localdelivery of therapeutic agents.

A therapeutic agent as used herein refers to an agent which canmitigate, cure, treat or prevent a disease or condition. It isparticularly desirable that the therapeutic agent be capable of exertingit effect locally (i.e., at or near the site of the disease orcondition). Exemplary therapeutic agents include, but are not limitedto, antibiotics, antirestenotics, anti-proliferative agents,anti-neoplastic agents, chemotherapeutic agents, cardiovascular agents,anti-inflammatory agents, immunosuppressive agents, anti-apoptotic andanti-tissue damage agents.

In the context of the present invention, an antibiotic is intended toinclude antibacterial, antimicrobial, antiviral, antiprotozoal andantifungal agents. Representative examples of such agents includeantibiotics such as aminoglycosides (e.g., streptomycin, gentamicin,tobramycin); 1st, 2nd, and 3rd generation cephalosporins (e.g.,cephalothin, cefaclor, cefotaxime, moxalactam, other semisyntheticcephalosporins such as cefixime); penicillins (e.g., penicillin G,ampicillin, amoxicillin); quinolones (e.g., ciprofloxacin, nalidixicacid, ofloxacin, tosufloxacin, lomefloxacin); sulfonamides (e.g.,sulfamethizole, sufisoxazole, sulfasalazine, trimethoprim);tetracyclines (e.g., doxycycline, methacycline); macrolides (e.g.,erythromycins); monobactams (e.g., aztreonam, loracarbef); andmiscellaneous agents such as novobiocin, rifampin, bleomycin,chloramphenicol, clindamycin, kanamycin, neomycin, spectinomycin,amphotericin B, colistin, nystatin, polymyxin B, cycloserine,methenamine, metronidazole, rifabutan, spectinomycin, trimethoprim,bacitracin, vancomycin, other β-lactam antibiotics. Antifungal agentsinclude flucytosine, fluconazole, griseofluvin, ketoconazole andmiconazole. Antiviral and AIDS agents include acyclovir, amantadine,didanosine (formerly ddI), griseofulvin, flucytosine, foscamet,ganciclovir, idoxuridine, miconazole, clotrimazole, pyrimethamine,ribavirin, rimantadine, stavudine (formerly d4T), trifluridine,trisulfapyrimidine, valacyclovir, vidarabine, zalcitabine (formerly ddC)and zidovudine (formerly AZT). Representative examples of antiprotozoalagents include pentamidine isethionate, quinine, chloroquine, andmefloquine.

Representative examples of restenosis therapeutic agents include, forexample, anti-angiogenic agents such as anti-invasive factor (Eisentein,et al. (1975) Am. J. Pathol. 81:337-346; Langer, et al. (1976) Science193:70-72; Horton, et al. (1978) Science 199:1342-1345), retinoic acidand derivatives thereof which alter the metabolism of extracellularmatrix components to inhibit angiogenesis, tissue inhibitor ofmetalloproteinase-1, tissue inhibitor of metalloproteinase-2,plasminogen activator inhibitor-1, plasminogen activator inhibitor-2,and anginex (Griffioen, et al. (2001) Biochem. J. 354(Pt 2):233-42);collagen inhibitors such as halofuginone or batimistat; antisenseoligonucleotides directed to nucleic acid sequences encoding c-myc orc-myb; growth factor inhibitors such as tranilast, trapidil orangiopeptin; antioxidants such as probucol, anti-thromobotics such asheparin or abciximab, anti-proliferative agents such as AG-1295(Fishbein, et al. (2000) Arterioscler. Thromb. Vasc. Biol. 20:667),tyrphostin (Banai, et al. (2005) Biomaterials 26(4):451-61), pacitaxelor other taxanes (Scheller, et al. (2004) Circulation 110(7):810-4),isoflavones (Kanellakis, et al. (2004) Atherosclerosis 176(1):63-72),rapamycin or derivatives or analogs thereof (Schachner, et al. (2004)Ann. Thorac. Surg. 77(5):1580-5), vincristine, vinblastine, HMG-CoAreductase inhibitors, doxorubicin, colchicines, actinomycin D, mitomycinC, cyclosporine, or mycophenolic acid; anti-inflammatory agents such asdexamethasone (Liu, et al. (2004) Expert Rev. Cardiovasc. Ther.2(5):653-60), methylprednisolone, or gamma interferon; and the likewhich exhibits antirestenotic activity.

Other therapeutic agents that can be utilized in accordance with thepresent invention include anti-proliferative, anti-neoplastic orchemotherapeutic agents to prevent or treat tumors. Representativeexamples of such agents include androgen inhibitors; antiestrogens andhormones (e.g., flutamide, leuprolide, tamoxifen, estradiol,estramustine, megestrol, diethylstilbestrol, testolactone, goserelin,medroxyprogesterone); cytotoxic agents (e.g., altretamine, bleomycin,busulfan, carboplatin, carmustine(BiCNU), cisplantin, cladribine,dacarbazine, dactinomycin, daunorubicin, doxorubicin, estramustine,etoposide, lomustine, cyclophosphamide, cytarabine, hydroxyurea,idarubicin, interferon alpha-2a and -2b, ifosfamide, mitoxantrone,mitomycin, paclitaxel, streptozocin, teniposide, thiotepa, vinblastine,vincristine, vinorelbine); antimetabolites and antimitotic agents (e.g.,floxuridine, 5-fluorouracil, fluarabine, interferon alpha-2a and -2b,leucovorin, mercaptopurine, methotrexate, mitotane, plicamycin,thioguanine, colchicines); folate antagonists and otheranti-metabolites; vinca alkaloids; nitrosoureas; DNA alkylating agents;purine antagonists and analogs; pyrimidine antagonists and analogs;alkyl solfonates; enzymes (e.g., asparaginase, pegaspargase); and toxins(e.g., ricin, abrin, diphtheria toxin, cholera toxin, gelonin, pokeweedantiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin′-A).

Further therapeutic agents that can be utilized within the presentinvention include cardiovascular agents such as antihypertensive agents;adrenergic blockers and stimulators (e.g., doxazosin, guanadrel,guanethidine, pheoxybenzamine, terazosin, clonidine, guanabenz);alpha-/betα-adrenergic blockers (e.g., labetalol); angiotensinconverting enzyme (ACE) inhibitors (e.g., benazepril, catopril,lisinopril, ramipril); ACE-receptor antagonists (e.g., losartan); betablockers (e.g., acebutolol, atenolol, carteolol, pindolol, propranolol,penbatolol, nadolol); calcium channel blockers (e.g., amiloride,bepridil, nifedipine, verapamil, nimodipine); antiarrythmics, groupsI-IV (e.g., bretylium, lidocaine, mexiletine, quinidine, propranolol,verapamil, diltiazem, trichlormethiazide, metoprolol tartrate, carteololhydrochloride); and miscellaneous antiarrythmics and cardiotonics (e.g.,adenosine, digoxin, caffeine, dopamine hydrochloride, digitalis).

Other therapeutic agents that can be used in accord with the presentinvention include anti-inflammatory agents. Representative examples ofsuch agents include nonsteroidal agents (NSAIDS) such as salicylates,diclofenac, diflunisal, flurbiprofen, ibuprofen, indomethacin, mefenamicacid, nabumetone, naproxen, piroxicam, ketoprofen, ketorolac, sulindac,tolmetin. Other anti-inflammatory drugs include steroidal agents such asbeclomethasone, betamethasone, cortisone, dexamethasone, fluocinolone,flunisolide, hydorcortisone, prednisolone, and prednisone.Immunosuppressive agents are also contemplated (e.g.,adenocorticosteroids, cyclosporin).

Other therapeutic agents include anti-tissue damage agents.Representative examples of such agents include superoxide dismutase;immune modulators (e.g., lymphokines, monokines, interferon α and β);and growth regulators (e.g., IL-2, tumor necrosis factor, epithelialgrowth factor, somatrem, fibronectin, GM-CSF, CSF, platelet-derivedgrowth factor, somatotropin, rG-CSF, epidermal growth factor, IGF-1).

In a particular embodiment, the therapeutic agent is an anti-restenoticagent such as rapamycin (i.e., sirolimus) or a derivative or analogthereof, e.g., everolimus or tacrolimus (Grube, et al. (2004)Circulation 109(18):2168-71; Grube and Buellesfeld (2004) Herz29(2):162-6).

In another embodiment, the therapeutic agent is an anti-apoptotic agentsuch as Galectin-3; (−)deprenyl; monoamine oxidase inhibitors (MAO-I)such as selegiline and rasagiline; Rapamycin; or querceten.

In general, the therapeutic agent can be added concurrent with orsubsequent to the preparation of the nanoparticles. The therapeuticagent is desirably loaded into preformed nanoparticles with loading ofat least 3% w/w of agent to nanoparticles. Generally, it is desirable toachieve loading of up to 10% w/w of therapeutic agent to nanparticle.

The present invention further relates to a method for preventing ortreating a disease or condition using the nanoparticles disclosedherein. The method involves locally administering an effective amount ofa composition containing a therapeutic agent encapsulated in ananoparticle composed of an N-alkylacrylamide, a vinyl monomer, and aPEG conjugate to a patient having or at risk of a disease or conditionthereby preventing or treating the disease or condition in the patient.

A patient having a disease or condition, in general, exhibits one ormore signs associated with the disease or condition. A patient at riskof a disease or condition is intended to include a patient that has afamilial history of the disease or condition or due to othercircumstances may be predisposed to develop the disease or condition.For example, a patient at risk of developing restenosis would include apatient that has undergone intervention to relieve an arterialobstruction (e.g., angioplasty, atherectomy, or stenting) and may be atrisk of developing stenosis. When delivered locally (e.g., at the siteof injury or at the site of a tumor), a composition of the presentinvention can deliver a sustained-release of the therapeutic agent toprevent or treat a select disease or condition. In general, an effectiveamount is considered an amount that causes a measurable change in one ormore signs or symptoms associated with the select disease or conditionwhen compared to otherwise same conditions wherein the agent is notpresent. For example, an effective amount of an anti-proliferative agentwould cause a measurable decrease in hyperplasia or cell proliferationas compared to cells not exposed to the anti-proliferative agent.Further, an effective amount of an antibiotic would result in aninhibition or decrease in the number of viable bacterial, fungal, orprotozoan cells.

Nanoparticle compositions of the present invention can be administeredeither alone, or in combination with a pharmaceutically orphysiologically acceptable carrier, excipient or diluent. Generally,such carriers should be nontoxic to recipients at the dosages andconcentrations employed. Ordinarily, the preparation of suchcompositions entails combining the nanoparticle composition of thepresent invention with buffers, antioxidants such as ascorbic acid, lowmolecular weight (less than about 10 residues) polypeptides, proteins,amino acids, carbohydrates including glucose, sucrose or dextrins,chelating agents such as EDTA, glutathione and other stabilizers andexcipients.

As demonstrated herein, local drug delivery facilitates high regionalconcentrations of the therapeutic agent with prolonged retention atlower doses with reduced systemic toxicity. In addition, therapeuticagents with a relatively short half-life, such as recombinant proteinsand peptides, and other biologically unstable biomolecules such asnucleic acids and oligonucleotides, can also be delivered locally withminimal loss in therapeutic activity before uptake by the target cellsor tissue. Furthermore, the hydrophobic core of the nanoparticlecomposition of the present invention will find use in the encapsulationand delivery of highly hydrophobic therapeutic agents. Moreover, localdrug delivery reduces patient-to-patient variability in drugpharmacokinetics, which is usually associated with intravenous or oralroutes of drug administration.

Nanoparticle compositions provided herein can be prepared for localadministration by a variety of different routes, including for example,directly to site of the disease or condition (e.g., a site of injury ortumor) under direct vision (e.g., at the time of surgery or viaendoscopic procedures) or via percutaneous drug delivery to the exterior(adventitial) surface of the site of the disease or condition (e.g.,perivascular delivery). As an alternative, the placement of pellets viaa catheter or trocar can also be accomplished.

Perivascular drug delivery involves percutaneous administration of thenanoparticle composition using a needle or catheter directed viaultrasound, computed tomography, fluoroscopic, positron emissiontomography, magnetic resonance imaging or endoscopic guidance to thesite of the disease or condition. Alternatively, the procedure can beperformed intra-operatively under direct vision or with additionalimaging guidance. In the case of restenosis or other cardiovasculardiseases, such a procedure can also be performed in conjunction withendovascular procedures such as angioplasty, atherectomy, or stenting orin association with an operative arterial procedure such asendarterectomy, vessel or graft repair or graft insertion.

For example, in a patient with narrowing of the superficial femoralartery, balloon angioplasty would be performed in the usual manner(i.e., passing a balloon angioplasty catheter down the artery over aguide wire and inflating the balloon across the lesion). Prior to, atthe time of, or after angioplasty, a needle would be inserted throughthe skin under ultrasound, fluoroscopic, or CT guidance and atherapeutic agent (e.g., rapamycin encapsulated into a sustained-releasenanoparticle) would be infiltrated through the needle or catheter in acircumferential manner directly around the area of narrowing in theartery. This could be performed around any artery, vein or graft, butideal candidates for this intervention include diseases of the carotid,coronary, iliac, common femoral, superficial femoral and poplitealarteries and at the site of graft anastomosis. Logical venous sitesinclude infiltration around veins in which indwelling catheters areinserted.

Those of ordinary skill in the art can readily identify the appropriatetherapeutic agent for the prevention or treatment of a select disease orcondition and optimize effective doses and co-administration regimens asdetermined by good medical practice and the clinical condition of theindividual patient. Regardless of the manner of administration, it canbe appreciated that the actual preferred amounts of active agent in aspecific case will vary according to the particular formulation and themanner of administration. The specific dose for a particular patientdepends on age, body weight, general state of health, on diet, on thetiming and route of administration, and on medicaments used incombination and the severity of the particular disorder to which thetherapy is applied. Dosages for a given subject can be determined usingconventional considerations, e.g., by customary comparison of thedifferential activities of the selected agent and of a known agent, suchas by means of an appropriate conventional pharmacological protocol.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1 Formulation of Rapamycin-Loaded Nanoparticles

N-Isopropylacrylamide (NIPAM) was purchased from Sigma Chemical Co (St.Louis, Mo.) and purified by recrystallization from n-hexane beforepolymerization. N-Vinyl pyrrolidone (VP) was obtained from Fluka ChemieAG and RdH (Laborchemikalien GmbH & Co. KG) and was distilled beforeuse. N,N′-Methylene bis-acrylamide (MBA), ferrous ammonium sulfate(FAS), ammonium persulphate (APS), TWEEN®-80, and rapamycin werepurchased from Sigma (St. Louis, Mo.). All salts used in the preparationof buffers were from Fisher Scientific (Pittsburgh, Pa.). All aqueoussolutions were prepared with distilled and deionized water (Water proplus, Labconco, Kansas City, Mo.).

Nanoparticles were formulated through random, free radicalpolymerization. In brief, water-soluble monomers NIPAM, VP and PEGylatedmaleic ester were used in a various molar ratios and then cross-linkedwith MBA. FAS was used to activate the polymerization reaction. In atypical optimized protocol, 19 μL of freshly distilled VP, 80 mg ofNIPAM, and 10 mg of PEGylated maleic ester were dissolved in 10 mLwater. To this aqueous solution was added 28 μL of MBA (49 mg/mL) andnitrogen gas was passed through the solution for 30 minutes to removedissolved oxygen. Subsequently, 50 μL of 10% FAS and 50 μL of saturatedAPS solution were added to initiate the polymerization reaction. Thepolymerization reaction was carried out at 30° C. under nitrogenatmosphere for 24 hours. The nanoparticle dispersion thus formed wasdialyzed overnight against distilled water (2 L) using a SPECTROPORE®dialysis bag (molecular weight cutoff 12-kD, SPECTRUM®, Laguna Hills,Calif.,) for 24 hours with water changed twice to remove unreactedmonomers and electrolytes. The aqueous dispersion of nanoparticles waslyophilized (−80° C., <10 μm mercury pressure, SENTRY™, Virtis,Gardiner, N.Y.) for 48 hours to obtain a dry power, which wassubsequently used for drug loading.

For rapamycin loading, 20 mg of the lyophilized nanoparticles wasdispersed in 2 mL of distilled water by vortexing for 2 minutes. To thisdispersion was added 250 μL of methanolic solution of rapamycin (4mg/mL) with constant stirring on a magnetic stir plate for 2 hours. Thisallowed rapamycin to partition into the hydrophobic core of thenanoparticles. The free rapamycin was separated by overnight dialysis ofthe dispersion against 1 L of distilled water using a SPECTROPORE®dialysis bag (molecular weight cutoff size 12-kD). The drug-loadednanoparticles were then lyophilized for 48 hours as described herein.For arterial localization of nanoparticles, the formulation contained afluorescent dye, 6-coumarin. The dye solution (100 μL, 0.5 mg/mL) wasadded into the micellar dispersion instead of a drug solution.Localization of the dye, and hence the nanoparticles was carried outconfocal microscopy.

The resulting nanoparticles were characterized by an ¹H NMR spectra ofmonomers and polymers recorded on Varian 500 MHz spectrophotometer.Nanoparticles were dissolved in D₂O to demonstrate that thepolymerization was complete. Particle size distribution (mean diameterand poly-dispersity index) of the nanoparticles, prior to and after drugloading was determined by photon correlation spectroscopy usingquasi-elastic light scattering equipment (ZETAPLUS™ particle sizeanalyzer, Brookhaven Instrument Corp., Holtsville, N.Y.) and ZETAPLUS™particle size software (Version 2.07). To measure particle size, adilute dispersion of nanoparticles in HEPES buffer (0.1 mg/mL, 0.001 MpH 7.0) was prepared. The same sample was used to measure zeta potentialof particles using ZETAPLUS™. Particle size of nanoparticles was alsodetermined by transmission electron microscopy (TEM). A drop ofrapamycin-loaded nanoparticles in water was placed on a FORMVAR®-coatedcopper grid, followed by a negative staining with 2% (w/v) uranylacetate solution. Particles were visualized using a Philips 201 TEMmicroscope (Philips/FEI Inc., Briarcliff Manor, N.Y.).

The ¹H NMR spectra of the copolymer demonstrated the absence of vinylend group protons of the monomers, indicating that polymerization wascomplete. The mean hydrodynamic diameter of nanoparticles was ˜70 nmwith a narrow size distribution (polydispersity index=0.11) and zetapotential (surface charge) of −8.45 mV at pH 7. The particle size ofmicellar nanosystem increased slightly (mean diameter ˜70 vs. ˜76 nm)following drug loading. The nanoparticles formed a colloidal dispersionin phosphate buffered saline (PBS). TEM of the nanoparticlesdemonstrated almost spherical shape, with a mean diameter of 61±7 nm(mean±SD; n=20). The particle size obtained with TEM is smaller thanthat measured with laser light scattering because the latter measureshydrodynamic diameter that includes hydration of the PEG at the outerlayer of the nanoparticle. The drug loading was 4.2% w/w (i.e., 100 mgof formulation contained 4.2 mg of rapamycin); with an encapsulationefficiency of 84% (i.e., 84% of the added drug was trapped innanoparticles).

Example 2 Drug Release from Nanoparticles

Release of rapamycin from nanoparticles in vitro was determined in PBS(154 mM, pH 7.4) containing 0.1% w/v TWEEN®-80 to maintain the sinkcondition. The donor chamber of each cell was filled with a 2.5 mLdispersion of nanoparticles (2 mg/mL) in buffer and the receiver chamberwas filled with the same buffer. A MILLIPORE® membrane with 0.1 μm poresize (Millipore Co., Bedford, Mass.) was placed between the twochambers. The cells were placed on a shaker maintained at 37° C. androtated at 100 rpm (ENVIRON®, Lab Line, Melrose Park, Ill.). Atpredetermined time intervals, the solution from the receiver side wascompletely removed and replaced with fresh buffer. Rapamycinconcentration in the collected samples was determined by HPLC (ShimadzuScientific Instrument, Inc., Columbia, Md.). The mobile phase consistingof methanol: water (9:1 v/v) delivered at a flow rate of 0.4 mL/minute(pump Model LC-10AT). A 20 μL of sample was injected by an autoinjector(Model SIL-10A) and the separations were achieved using a NOVA-PARK® C-8column (2×150 mm², 4 μm size packing; Phenomenex, Torrance, Calif.).Rapamycin levels in the samples were quantified by UV detection (A=276nm, Model SPD-10A VP, Shimadzu). A standard plot of rapamycin (0-50μg/mL) was prepared under identical conditions.

The release profile of rapamycin from the nanoparticles disclosed hereinunder in vitro conditions demonstrated a relatively rapid drug releaserate during the initial stages (˜20% release in 24 hours) with moresustained release thereafter (more than 80% release in 28 days).

Example 3 Anti-Proliferative Effects of Rapamycin-Loaded Nanoparticles

Human vascular smooth muscle cells (Cascade Biologics, Portland, Oreg.)were maintained on medium 231 supplemented with smooth muscle growthsupplement (Cascade Biologics) at 37° C. in a humidified, 5% CO₂atmosphere. Cells passaged 3 to 4 times were typically used.

To monitor cell proliferation, cells were seeded at a 5,000 cell perwell density in 96-well plates and allowed to attach for 24 hours.Different doses of rapamycin (1 ng/mL to 1,000 ng/mL) either loaded innanoparticles or in solution (rapamycin dissolved in methanol wasdiluted in the medium) were used. The concentration of methanol in themedium was kept below 0.1% so that it had no effect on cellproliferation. Cells treated with empty nanoparticles or medium servedas respective controls for drug-loaded nanoparticles or drug insolution. The medium in the wells was changed on day two and on everyalternate day thereafter with no further addition of drug.Anti-proliferative activity of the drug was monitored for eight days ofthe study using an MTS assay (CELLTITER 96® AQ_(ueous), Promega,Madison, Wis.). MTS assay reagent (20 μL/well) was added to each welland the plates were incubated for 3 hours at 37° C. in a cell cultureincubator. Color intensity was measured at 490 nm using a microplatereader (Bio-Tek Instrument, Winooski, Vt.).

For cell cycle analysis, cells were seeded into T-75 culture flasks at acell density of 1×10⁶ cells per flask in 10 mL growth medium and wereallowed to attach overnight. The medium from each flask was replacedwith a dispersion of rapamycin-loaded nanoparticles in growth medium(dose of rapamycin=50 ng/mL). Two days following treatment, cellmonolayers were washed with PBS and the cells were detached bytrypsinization. DNA analysis was performed by staining cells withpropidium iodide, a fluorescent dye which intercalates between DNA basepairs. The cells were fixed with 70% ethanol, incubated for 1 hour, and1 mL of Telford reagent was added to the cell suspension. The cellularDNA content was analyzed by a fluorescent activated FACSTARPLUS® flowcytometer operating under Lysis II (Becton Dickinson ImmunocytometrySystems, San Jose, Calif.).

Example 4 Balloon Injury and Local Delivery in Rat Carotid Artery

Male Sprag-Dawley rats (240 to 260 grams; Charles River Laboratories,Wilmington, Mass.) were anesthetized with an intraperitoneal injectionof a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg). Through amidline neck incision, the left common, external and internal carotidarteries were exposed by blunt dissection. A 2F Fogarty balloon catheter(Edwards Life Sciences, Irvine, Calif.) was introduced in the leftexternal carotid artery via an arteriotomy and was advanced to theorigin of the left common carotid artery. The balloon was inflatedsufficiently to generate slight resistance and was withdrawn three timesconsistently to produce endothelial denudation of the entire length ofthe left common carotid artery. Upon removal of the balloon catheter, aPE 10 catheter was inserted into the left common carotid artery. The midand the distal portions of the left common carotid artery and the leftinternal carotid artery were temporarily tied off. A suspension ofrapamycin-loaded nanoparticles (200 μL containing 60 μg of rapamycinequivalent nanoparticles) was infused into the injured carotid arteryover 5 minutes at 2 atm of pressure (three, one-minute periods betweeninfusions of 70 μL of the suspension, with a one minute period betweeninfusions). Following infusion of the nanoparticles, the ties wereremoved and the blood flow was restored. In another group of animals,the same dose of rapamycin-loaded nanoparticles was injectedintraperitoneal to demonstrate that the effect of rapamycin oninhibition of restenosis is due to local drug delivery.

Example 5 Arterial Localization of Nanoparticles

To determine localization of nanoparticles in the layers of arterialwall (Intima, Media or Adventitia), particularly with time afterinfusion, a formulation of nanoparticles containing 6-coumarin dye wasinfused following balloon injury as described. The physical properties(particle size and zeta potential) of the dye-loaded nanoparticles weresimilar to the drug-loaded nanoparticles. At one hour and 24 hoursfollowing infusion of nanoparticles, the arteries were removed, rinsed,and embedded in O.C.T. compound (Tissue-Tek, Sakura, Torrance, Calif.)and stored in dark at −70° C. until histological evaluation. The frozenblocks were sectioned using a rotary microtome (AO 820, AmericanOptical, Del Mar, Calif.) and viewed with a confocal microscope. Theimages were captured using a 488-nm filter (Fluorescein), 568-nm filter(Rhodamine), and differential interference contrast using a ZeissConfocal microscope LSM410 equipped with argon-krypton laser (Carl ZeissMicroimaging, Thornwood, N.Y.).

To determine arterial uptake and drug retention, carotid arteries fromboth sides were removed at different time points followingadministration of rapamycin-loaded nanoparticles, arteries were rinsedwith saline and blotted dry using an absorbent paper. Each artery wasweighed (wet weight), finely cut into small pieces with a scissor,homogenized in 2 mL of distilled water using a tissue homogenizer(Biospace Product Inc, Bartlesville, Okla.) at 1,000 rpm for twominutes, and homogenates were lyophilized for 48 hours. Drug from eachdry tissue was extracted by shaking each sample with 1 mL methanol at37° C. for 48 hours at 150 rpm using an ENVIRON® orbital shaker. Thesamples were centrifuged at 14,000 rpm for 10 minutes (EPPENDORF®Microcentrifuge, 5417R, Brinkmann Instruments, Westbury, N.Y.) to removecell debris. The supernatant was analyzed by HPLC for rapamycin contentas described herein. A standard plot was prepared using arteriescollected from animals which did not receive rapamycin to determineefficiency of drug recovery.

Example 6 Inhibition of Restenosis

After three weeks, rats were anesthetized with an intraperitonealinjection of a mixture of ketamine (80 mg/kg) and xylazine (5 mg/kg).After the intravascular system was cleared, pressure fixation wasperformed by infusing 10% formaldehyde solution over 5 minutes at 120 mmHg. Left carotid arteries were retrieved and immersed in the samefixative until sectioned. The arteries were cut into pieces every 3 mmfrom proximal to distal ends. These pieces of arteries were embedded inparaffin for sectioning, and duplicate slides were stained withhematoxylin-eosin. The medial and intimal areas and luminal area weremeasured with a computerized digital image analysis system.

For immunohistochemical analysis, samples were incubated with I-VIEWinhibitor to block endogenous peroxidase activity. After washings inPBS, sections were incubated with primary antibody for one hour at roomtemperature. The following primary antibodies were used: monoclonalmouse 1A4 antibody recognizing α-SM actin (neat, DAKO, Carpenter,Calif.) and monoclonal mouse PC10 antibody for identifying PCNA (1:25dilution, DAKO, Carpenter, Calif.) and CD31 antibody (1:100 dilution,DAKO, Carpenter, Calif.) for endothelial staining, anti-cleavedcaspase-3 (1:200 dilution, Cell Signaling Technology, Beverly, Mass.)apoptotic cells by terminal deoxynucleotidyl transferases (TdT)-mediateddUTP nick end-labeling (TUNEL) method using TUNEL system kit (Promega,Madison, Wis.). Sections were subsequently incubated with I-VIEW biotinand I-VIEW streptavidin-horseradish peroxidase. Sections were visualizedusing DAB chromogen and were counterstained using I-VIEW copper. Thenumber of cells positive for PCNA and α-SM actin staining was counted ata magnification 400×. Endothelization was calculated as the ratiobetween the luminal surface covered by CD31 positive cells and the totalluminal surfaces.

All the data are presented as mean±SEM. The statistical significance ofdifferences between the untreated and treated groups was determined by aone-way ANOVA. Differences were considered significant if p<0.005.

What is claimed is:
 1. A sustained-release nanoparticle compositioncomprising a copolymer of an N-alkylacrylamide, a vinyl monomer, and apolyethylene glycol conjugate.
 2. The sustained-release nanoparticlecomposition of claim 1, further comprising a therapeutic agent.
 3. Amethod for preventing or treating a disease or condition comprisinglocally administering to a patient having or at risk of having a diseaseor condition, an effective amount of a composition of claim 2 therebypreventing or treating the disease or condition in the patient.